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G E Wu

Publications and source records attributed to G E Wu.

At least 19 recordsLinked to original sources
PubMed1992

Enumeration and characterization of DJH structures in mouse fetal liver.

The primary immunoglobulin (Ig) repertoire in the mouse develops during fetal life in the liver. The first Ig gene rearrangement--the joining of a DH to a JH gene segment--contributes largely to the diversity found in CDR3, as well as potentially encoding the D mu protein which is believed to function in the development of a B cell. In this report, the number of DJH joins in two mouse strains, C57BL/6 and BALB/c, were enumerated from days 12 to 16 of fetal development. It was found that the number of DJH structures increased from less than 300 per liver on day 12 to greater than 700,000 (C57BL/6) and 300,000 (BALB/c) on day 16. Each JH gene segment was used approximately equally on each day examined. When the DJH structures were examined by cloning and sequencing it was found that the DJH reading frame (RF) usage (with respect to JH) was not random--RF1 was used 70% of the time. Moreover, a single D gene segment, DFL16.1, was used in greater than 50% of all joins reinforcing the notion that the fetal repertoire is restricted in its antigen binding potential.

Animals↗
PubMed1991

Mouse kappa light-chain recombination signal sequences mediate recombination more frequently than do those of lambda light chain.

Immunoglobulin and T-cell receptor genes are somatically rearranged by site-specific recombination. Recombination signal sequences (RSS) have been identified as the major targeting element of this process. Recent reports demonstrate that differences in RSS affect the frequency of recombination, suggesting a role for RSS in the development of the B-cell repertoire. Examination of mouse light-chain RSS indicates that kappa light-chain RSS consistently show a greater degree of similarity to a consensus sequence than do those of lambda light chain. To determine whether this difference in natural RSS could affect the patterns of light-chain gene rearrangement and expression, we have constructed recombination substrates containing both a typical mouse kappa RSS pair and a typical mouse lambda RSS pair. Experiments using these substrates demonstrate that the kappa RSS pair mediates recombination at a vastly higher frequency than does the lambda RSS pair. This result argues that RSS differences may contribute significantly to the patterns of mouse immunoglobulin light-chain rearrangement, ultimately resulting in a high proportion of kappa light chain relative to lambda.

Animals↗
PubMed1991

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers.

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.

Abelson murine leukemia virus↗
PubMed1991

Measurement of recombinase activity in a set of related Abelson murine leukemic virus pre-B cell lines: DJ/DJ lines have more recombinase activity than do VDJ/VDJ lines.

The VDJ recombination potential of a number of Abelson murine leukemic virus transformed fetal liver cell lines derived from (C57BL/6 x BALB/c) F1 mice was measured. The specific developmental stage of each line was determined using Southern blot analysis to ascertain their rearrangement status at the immunoglobulin heavy chain locus (DJ/DJ, VDJ/DJ or VDJ/VDJ). It was observed that DNA from DJ/DJ lines gave many more 'subhaploid' bands hybridizing with JH than did DNA from VDJ/VDJ lines. While the lack of appropriate substrate (VDJ/VDJ lines have exhausted the normal IgH substrate) contributes to the decrease in 'subhaploid bands', this result indicates that the rate of ongoing immunoglobulin heavy chain gene rearrangement was higher in the DJ/DJ lines. However, when the lines were examined using the assay developed by Hesse et al. (4) to measure VDJ recombinase activity, it was found that although all lines had recombinase activity, the DJ/DJ lines had four times more VDJ recombinase activity than did the VDJ/VDJ lines.

Abelson murine leukemia virus↗
PubMed1991

B cell commitment.

Multipotential stem cells have the capacity to differentiate into a variety of cell types including B lymphocytes. The development of B cells from multipotential progenitors has been the subject of numerous studies and many aspects of this transition are well characterized. However, the initial genetic events which commit a stem cell to follow the B lineage developmental pathway have not yet been discovered. In this review we examine diverse experiments which may lead to increased understanding of the important first steps in the commitment process.

Animals↗
PubMed1990

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein.

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.

Animals↗
PubMed1990

Characterization of a 70Z/3 pre-B cell derived macrophage clone. Differential expression of Hox family genes.

An adherent cell line was established from the much studied pre-B cell line 70Z/3. The adherent cell line had the morphological features of a macrophage and stained positive for non-specific esterase. In addition, this line expressed high levels of RNA for the macrophage specific enzyme, lysozyme. Although the 70Z/3 macrophage variant has lost the ability to respond to lipopolysaccharide and interferon-gamma by expressing RNA transcripts for immunoglobulin light chain, it exhibited a new response characterized by increased levels of I-A RNA transcripts. Both the pre-B and macrophage variants also expressed RNA transcripts which hybridize to a Hox 2.3 probe. In contrast, transcripts which hybridize to Hox 1.1, 2.1, and 6.1 probes are expressed in the pre-B line but could not be detected in the macrophage.

Animals↗
PubMed1990

VH gene repertoire.

In this review, we have assembled some results on VH gene usage by mouse and human. We conclude that there is an early bias in usage of certain VH genes in both mouse and human. This biased usage has a strain dependent component as evidenced by its continued presence in the adult repertoire of some mouse strains, notably BALB/c, and not in others. The reason for the fetal bias is uncertain. However, the finding that the VH gene segments used in the human fetal repertoire are similar in sequence but not in chromosomal position to those expressed in the mouse fetal-repertoire leads us to suggest that the bias is not due to chromosomal location but rather may be reflecting the functioning of these gene products early in ontogeny.

Animals↗
PubMed1989

A linkage map of the mouse immunoglobulin lambda light chain locus.

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes. The order of the lambda gene segments is V2-J2C2J4C4-V1-J3C3J1C1, as was originally proposed. No evidence for nonmethylated CpG rich areas (HTF islands) within the region was found. Fine mapping using the lambda 1, lambda 3 rearranged cell line J558 mapped the gap between the V and J-C gene segments in the lambda 1 gene cluster (V1-J3C3J1C1) to approximately 70 kb. The similar distance (60-100 kb) found in the lambda 2 gene cluster (V2-J2C2J4C4) is further evidence that duplication of an ancestral locus occurred.

Animals↗
PubMed1989

The B cell repertoire.

The hallmark of the immune system is its ability to produce a seemingly infinite variety of antigen-binding receptors. This is made possible by molecular and cellular mechanisms uniquely suited to continuously generate a large number of individual receptor molecules and to select some for further expansion. The well-studied genetic rearrangement that results in the juxtaposition of germ line-encoded variable, diversity, and joining elements remains the foundation for diversification on which the repertoire is built. Many of the rules that regulate this phenomenon have been described, although the underlying enzymatic machinery responsible for these events remains to be elucidated. Recent progress in categorizing the immunoglobulin heavy-chain variable region genes into families as well as studies establishing their utilization in both fetal and adult life is helping to further refine these rules. Subsequent cellular interactions 1) permit the discriminant expansion of clones expressing relevant antibody molecules, 2) allow the active affinity alterations needed for effective ongoing immune responses, and 3) limit the potential deleterious effect of autoreactive cells.

Animals↗
PubMed1988

VH gene family utilization is regulated by a locus outside of the VH region.

We have used the RNA colony blot method to examine VH usage in colonies derived from primary splenic B cells. We found that there are strain-specific differences in the pattern of VH usage. Using parental F1, congenic, and recombinant inbred strains we demonstrate that the genetic element that causes the observed phenotype is: (a) stably expressed; (b) not due to maternal influence; (c) not due to dominate diffusible factors; (d) not linked to cloning efficiency; and (e) outside the Igh locus.

Animals↗
PubMed1987

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.

Animals↗
PubMed1986

VH gene family utilization in colonies derived from B and pre-B cells detected by the RNA colony blot assay.

The immunoglobulin heavy chain variable region (VH) genes of the mouse have been categorized into families based upon sequence homology. Utilizing the RNA colony blot assay we have determined the expression of eight of these families in B cell colonies derived from either surface immunoglobulin positive (sIg+) adult spleen B cells or sIg- fetal liver pre-B cells. We demonstrate, based upon the analysis of greater than 6000 individual colonies, that VH gene usage is a characteristic of the mouse strain studied. C57BL/6 mice most frequently (45%) utilize family VHJ558, the largest VH family, whereas BALB/c mice most frequently (22%) utilize family VH7183, the most JH proximal family in BALB/c mice. Moreover, colonies derived from sIg- fetal liver derived precursors show similar patterns, suggesting that selection based on exogenous antigen is not an important parameter in determining VH gene family usage.

Animals↗
PubMed1986

Detection of RNA transcripts in normal lymphoid and myeloid colonies.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

B-Lymphocytes↗
PubMed1986

A region of the immunoglobulin-mu heavy chain necessary for forming pentameric IgM.

In order to define the molecular requirements for IgM pentamer formation, we have isolated several mutant hybridomas which produce predominantly monomeric IgM. For one such mutant, 102, we synthesized a cDNA clone of its mu-mRNA, and found an in-frame 39-bp deletion, which thus encodes a mu-chain lacking amino acids 550-562, a region spanning the fourth constant domain and the tail of the mu-chain. To prove that this deletion is sufficient to block pentamer formation, we used site-directed mutagenesis to construct a mu-gene lacking these 39 bp, and have shown that the expression of this altered mu-gene results in the production of monomeric IgM.

Amino Acid Sequence↗